Rotavirus is the leading cause of viral gastroenteritis in infants and piglets (2, 3, 4, 7, 8, 9, 17, 20, 27, 37). Rotaviruses, which are found in a great variety of animal species, are named for their characteristic wheel-like appearance under the electron microscope. Like other Reoviridae, their genome is in the form of double-stranded (ds) RNA, although they may be distinguished from reoviruses and orbiviruses by the division of their genome into 11 ds RNA segments.
In 1983, Pedley (24) classified the rotaviruses into several types of groups on the basis of serological differences, immunofluorescence, and nucleic acid differences characterized by one dimensional terminal fingerprint analysis. RNA electropherotype has also been used as a basis for classification (29). Group A rotaviruses are considered "typical"; all others (B, C, D, E) are referred to as "atypical." Type-C (Group-C) rotaviruses, sometimes referred to as pararotavirus, have been found to produce gastroenteritis in chickens, pigs, and also in humans (1, 4, 5, 7, 10, 11, 12, 13, 14, 20, 21, 22, 25, 26, 33, 34). A panel of sixteen monoclonal antibodies have been made to the porcine Type-C rotavirus (Cowden strain), with four of these having neutralizing capabilities. Only Type-C antigens were detected by the monoclonal antibodies tested, and no reactions were seen with Type-A or Type-B rotaviruses (23).
Analysis of the structural polypeptides of porcine Type-C rotavirus by Western blot analysis has revealed that there is a lack of cross-reaction between the structural polypeptides of porcine rotaviruses Types A, supporting the fact that both are distinctly different from each other, and thus the different serogroup classification (6, 18, 23).
Type-C rotavirus has been implicated as a cause of diarrhea in nursing and weakling pigs (4, 8, 15, 17, 21). Diagnostic surveys, conducted over several years, have revealed that Type-C rotavirus infections are responsible for 25% of preweaning scours cases and 40% of postweaning scours cases where rotavirus was deemed to be the causative agent (17). In Australia, 7 out of 235 cases of rotavirus diarrhea were linked to Type-C rotavirus by gel electrophoresis (21).
There is additional evidence of Type-C rotavirus prevalence as measured by Type-C, specific serum antibody. In Ohio, 100% of adult pigs, 59% of weaning pigs, and 86% of nursing pigs showed exposure to Type-C rotavirus, as measured by serum antibody levels (Specified Indirect Immunofluorescence; IFA, 31). In the United Kingdom, 58-90% of piglets from three to twenty-six weeks old had positive Type-C antibody titers as measured by IFA, whereas 77% of adult swine showed previous exposure to Type-C by IFA (9, 11).
Porcine Type-C rotavirus has been found to cross-react with at least eight different isolates of Human Type-C rotavirus by Immune Electron Microscopy (IEM) and IFA serological assay, suggesting that one common group antigen exists between porcine and human Type-C rotavirus strains (7, 10, 12, 20, 25, 26, 27, 33, 34).
Type-A rotaviruses have been successfully propagated in several different cell lines, but they require incorporation of either proteolytic enzymes, DEAE dextran, or a combination of both. Use of increased virus inoculum volumes has also contributed to the success in growing some of the type-A human rotavirus strains (42). Type-A-Rotavirus diagnostic kits, and bovine and porcine Type-A Rotavirus modified live virus vaccines are commercially available (38, 39, 40, 15 16, 35, 36). Human vaccines have been developed but not commercialized.
Limited replication of porcine Type-C rotavirus (Cowden strain) has been demonstrated in two types of cell cultures: Primary Pig Kidney (PK) and embryonic Rhesus Monkey Kidney (Ma-104) (28, 30). The intestinal origin Type-C rotavirus was maintained in PK cells for 17 passages by incorporating high (cytotoxic) levels of proteolytic enzyme (pancreatin, 30) e.g., 80-120 .mu.g/ml. Pancreatin is a mixture of several enzymes consisting of proteases (e.g., trypsin, chymotrypsin, alpha trypsin, etc), lipases, and amylases. Type-C rotavirus PK pass-9 was used as the inoculum for subpassage in Ma-104's. Again, high levels of pancreatin were required for maintenance of the virus. At these high levels of pancreatin, viral cytopathic effect (CPE) was not readily observed, due to the cellular toxic effect (e.g., detachment of the cells) of the proteolytic enzymes on the cell cultures. (When only 40 .mu.g/ml pancreatin was used, virus growth ceased after 3 passages.) The PK-passaged Type-C rotavirus was then passaged in the Ma-104's eighteen times, resulting in a peak titer of 5.times.10.sup.6 fluorescent focus units/ml at the sixteenth passage.
However, none of the cell culture passes have been reported to contain virus titers higher than 5.times.10.sup.6 FFU/ml (Fluorescent Foci Units). In addition, the 22nd and 26th cell culture passages were fed to gnotobiotic pigs by these workers and were still found to be pathogenic. Animals developed diarrhea and demonstrated villious atrophy. Propagation of porcine Type-C rotavirus in Ma-104 cell cultures directly from intestinal contents of infected pigs was unsuccessful (28). Attempts by other laboratories to propagate Type-C rotaviruses in cell culture, using either the reported Type-C techniques or the previously reported techniques used in growth of Type-A's have been unsuccessful (28, 30, 31, 33).The use of primary tissue culture (PK) for growing viruses suffers from the disadvantage that the primary tissue cultures are prone to contamination with not easily detected viruses, and the probability of which cannot be thoroughly established prior to actual use of the primary tissue. Thus, vaccine production in primary tissues are susceptible to extraneous virus contaminations which may not be detected until well after the preparation of the vaccine component.
It is obvious from epidemiological studies in swine (8, 9, 17, 31) that there is a need for an effective vaccine be it either inactivated or attenuated. There is also a need for development of diagnostic aids for detecting Type-C rotavirus infections. A process of cultivating Type C rotavirus for numerous passages at high viral titer, preferably attenuating the virulence of the virus without substantial loss of immunogenicity, is therefore sought.
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